Dual-Stimuli Responsive 2D Supramolecular Organic Framework for the Detection of Azoreductase Activity

Article abstract of DOI:10.1002/chem.201904443

A 2D supramolecular organic framework (SOF) based on synthetic macrocycles has been constructed in water by a self-assembly strategy. Two new organic monomers of this SOF, possessing viologen and azobenzene functional groups, form a stimuli-responsive host–guest system upon cooperatively binding with cucurbit[8]uril rings. The reversible formation and dissociation of 2D SOF can be realized by the isomerization of azobenzene under ultraviolet and visible light. The light-responsive property of the SOF is highly reversible and stable for up to four cycles. Moreover, azoreductase produced by Escherichia coli can reduce the N=N double bond of azobenzene entities, resulting in fluorescence recovery of the system. As an excellent and effective fluorescent probe, the SOF can detect azoreductase activity for real-time monitoring of the growth process of Escherichia coli. The dual-stimuli responsive 2D SOF is envisioned to drive the development of responsive devices with complex functions.

Full text of DOI:10.1002/chem.201904443

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Accepted Article
Title: Dual-Stimuli Responsive 2D Supramolecular Organic Framework
for the Detection of Azoreductase Activity
Authors: Hao Zhang, Feng Liang, and Ying-Wei Yang
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DOI: 10.1002/((please add manuscript number))
Article type: Full Paper
Dual-Stimuli Responsive 2D Supramolecular Organic Framework for the Detection of
Azoreductase Activity
Hao Zhang, Feng Liang,* and Ying-Wei Yang*
H. Zhang, Prof. F. Liang, Prof. Y.-W. Yang
The State Key Laboratory of Refractories and Metallurgy, School of Chemistry and Chemical
Engineering, Wuhan University of Science and Technology, Wuhan 430081, P. R. China
*E-mails: feng_liang@whu.edu.cn; ywyang@jlu.edu.cn
H. Zhang, Prof. Y.-W. Yang
State Key Lab of Inorganic Synthesis and Preparative Chemistry, International Joint Research
Laboratory of Nano-Micro Architecture Chemistry (NMAC), College of Chemistry, Jilin
University, 2699 Qianjin Street, Changchun 130012, P. R. China
Keywords: cucurbit[n]uril, fluorescent probe, photoisomerization, stimuli-responsive system,
supramolecular organic framework
Abstract: A two-dimensional (2D) supramolecular organic framework (SOF) based on
synthetic macrocycles has been constructed in water by self-assembly strategy. Two new
organic monomers of this SOF, possessing viologen and azobenzene functional groups,
respectively, form a stimuli-responsive host-guest system upon cooperatively binding with
cucurbit[8]uril rings. The reversible formation and dissociation of 2D SOF can be realized by
the isomerization of azobenzene under ultraviolet and visible light. The light-responsive
property of the SOF is highly reversible and stable in four cycles. Moreover, azoreductase
produced by Escherichia coli can reduce the N=N double bond of azobenzene entities,
resulting in fluorescence recovery of the system. As an excellent and effective fluorescent
probe, the SOF can detect azoreductase activity for real-time monitoring of the growth
process of Escherichia coli. The dual-stimuli responsive 2D SOF is envisioned to drive the
development of responsive devices with complex functions.
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Introduction
Two-dimensional (2D) materials with distinctive planar structure and excellent mechanical
properties have received considerable attention in the fields of separation, sensing
recognition, electronic component, biomaterials, etc.[1-4] To satisfy the growing demand of 2D
materials, direct self-assembly has been introduced as a convenient and effective strategy for
large-scale preparation.[5-8] 2D supramolecular organic framework (SOF) is a typical and
popular kind of materials based on self-assembly strategy, which exhibits exceptional
properties and enormous application potentials.[9-11]
In the self-assembly process of 2D SOFs, disordered organic monomers are constructed
orderly to form a planar structure driven by supramolecular interactions, which not only
provide a powerful and highly directional driving force for self-assembly, but also further
stabilize the obtained 2D SOFs.[12-15] Different from covalent connections, supramolecular
interactions show more versatile chemical properties in a self-assembly system. Manifold
organic monomers are introduced into the 2D SOFs via inclusion-enhanced dimerization of
supramolecular macrocycles, enriching the variety of self-assembly systems.[16-17] On the
other hand, additional stimuli, such as pH, temperature, and redox, can reversibly break and
restore the dynamic balance of supramolecular interactions.[18-21] The reversibility of stimuli-
responsive supramolecular chemistry entrusts the attractive structures and prospective
applications of 2D SOFs in sensing, detection, and bio-related fields.[22-25]
Cucurbit[8]uril (CB[8]) has been confirmed as a common rigid macrocycle that can drive
the organic monomers to self-assemble into supramolecular polymers.[26-30] Viologen and
azobenzene derivatives can form ternary complexes with CB[8], which are well-documented
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supramolecular light-responsive systems based on the photoisomerism of azobenzene
derivatives.[31-32] Furthermore, azobenzene derivatives are extensively applied to fluorescent
probes since the system would exhibit a fluorescence recovery after reducing nitrogen double
bond.[33-35] CB[8] ternary complexes offer a general approach to construct highly complex
supramolecular structures and materials.[36-39]
Herein, we have designed and synthesized two special guest compounds, i.e., TPE-4MV
and AZO (Figure 1, Schemes S1-S3), which can form a dual-stimuli responsive 2D SOF
with CB[8] in water via cooperative self-assembly. Through light-mediated isomerization of
AZO, we have realized the reversible formation and dissociation of the 2D SOF.
Significantly, the SOF can detect in real-time the azoreductase activity of Escherichia Coli by
monitoring the enzymatic reduction of AZO.
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Figure 1. Schematic representation of the formation of a 2D supramolecular organic
framework (SOF) by the self-assembly of TPE-4MV, AZO, and CB[8], reversible formation
and dissociation realized through light-mediated isomerization, and azoreductase detection in
Escherichia Coli by the enzymatic reduction of AZO.
Results and Discussion
Compounds of TPE-4MV and AZO have multiple viologen and azobenzene functional
groups, respectively. The detailed synthesis was described in the supporting information. To
confirm the formation of the host-guest complex, TPE-4MV and AZO were mixed with
CB[8] in D2O one after another. As shown in the 1H NMR spectra (Figure S1, Supporting
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Information), obvious chemical shifts were observed after the addition of two guests,
indicating the complex formation in water. Moreover, the UV-Vis titration was performed,
and Job’s plots (Figure 2a,b) showed that the stoichiometry of TPE-4MV, CB[8], and AZO
was 1:4:2 in the host-guest complex, which proved the formation of these host-guest
complexes constructed by TPE-4MV, CB[8], and AZO in water.
Figure 2. (a) Job’s plot, determining the 1:4 stoichiometry of TPE-4MV and CB[8]. (b) Job’s
plot, suggesting the 1:4:2 stoichiometry of TPE-4MV, CB[8], and AZO. (c) DLS results of
AZO (0.1 mM), TPE-4MV (0.1 mM), and SOF (the concentration of TPE-4MV, CB[8], and
AZO in it was 0.1 mM, 0.4 mM, and 0.2 mM, respectively) in water at 298 K. (d) The SAXD
profile of the SOF solid (the concentration of TPE-4MV, CB[8], and AZO in it was 0.03 mM,
0.12 mM, and 0.06 mM, respectively).
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After the formation of the 2D SOF by mixing TPE-4MV, CB[8], and AZO at a molar
ratio of 1:4:2 in water has been confirmed, the properties of the SOF have been explored by
various testing methods including dynamic light scattering (DLS), small-angle X-ray
scattering (SAXD), atomic force microscopy (AFM), and scanning electron microscopy
(SEM). DLS results (Figure 2c) showed that the hydrodynamic diameter (DH) of AZO and
TPE-4MV was 0.63 and 1.29 nm, respectively. When CB[8] was added, the DH of the
complex increased rapidly to 190 nm, indicating that the organic monomers formed into large
assemblies in water. In addition, a scattering peak with d spacing of ca. 5.53 nm was clearly
noticed in SAXD profile (Figure 2d), suggesting the periodicity of the interior of the
assemblies.[40] All these results confirmed the SOF formation. On the other side, the
morphology of the SOF was investigated by SEM (Figure 3a) and AFM (Figure 3b). As
expected, a large number of sheet-like structures were observed, which validated the 2D
structure of the SOF. The thickness of the structures was ca. 32 nm, which was resulted from
the accumulation of the SOF during the drying process. The height of the structure was
further calculated by AFM and the obtained values (Figure 3d) were conformed to the outer
diameter of CB[8] (1.75 nm),[41] which strongly indicated the structure of the 2D SOF.
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Figure 3. (a) SEM image of the 2D SOF (the concentration of TPE-4MV, CB[8], and AZO in
it was 0.3 mM, 1.2 mM, and 0.6 mM, respectively.) obtained by evaporating the solution. (b)
Tapping-mode AFM image of the SOF (the concentration of TPE-4MV, CB[8], and AZO in it
was 0.03 mM, 0.12 mM, and 0.06 mM, respectively). (c) Schematic representation of the 2D
SOF and height analysis (d) along the red and blue line on the AFM image.
In a supramolecular self-assembly system, stimuli-responsive host-guest complexes are a
research focus of great interests and significance, which are applied in structuring logical
molecular devices and controlling material properties.[42] Generally, the complexes will be
dissociated when stronger guest or host molecules are added.[43-45] Meanwhile, the introduced
chemicals will cause system pollution. As a green and environment friendly method, light can
provide a stable, accurate, and long-range external stimulus and has become a more
appropriate and attractive option. AZO with azobenzene groups showed the trans-cis
isomerism under the irradiation of UV light (365 nm) and visible light (450 nm). Two isomers
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can be distinguished from the UV-Vis spectra (Figure S2, Supporting Information) and 1H
NMR spectra (Figure S3, Supporting Information). Based on the photoinduced
isomerization phenomenon, reversible formation and dissociation of the 2D SOF can be
regulated by UV light and visible light. As shown in Figure 4a, the absorption peak at 345
nm in the near-UV region of the SOF solution decreased, while the absorption peak at 432 nm
in the visible region increased, showing the rapid transition from trans-AZO to cis-AZO
under the UV light (365 nm) within 3 min (Figure S4, Supporting Information). It is
geometrically unsuitable for the structurally altered AZO to enter the cavity of CB[8],[46-47]
resulting in the disintegration of 2D SOF. This was further illustrated by SEM (Figure 4c),
which showed the disorderly structure of the SOF after UV irradiation.
Figure 4. (a) The UV-Vis spectra of 2D SOF (the concentration of TPE-4MV, CB[8], and
AZO in it was 0.01 mM, 0.04 mM, and 0.02 mM, respectively) after irradiation with UV light
(365 nm) and visible light (450 nm). (b) Absorbance changes at 345 nm during UV and
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visible irradiation for four cycles. And SEM images of (c) the light-induced dissociation and
(d) re-formation of the 2D SOF.
To prove the reassembly of the 2D SOF, the mixture was exposed to visible light (450
nm). The absorption peaks of the SOF showed slow recovery during 8 min (Figure S5,
Supporting Information), as shown in the UV-Vis spectra (Figure 4a). Meanwhile, the
chaotic mixture reshaped into the smooth and large-area planes, testifying the re-formation of
the 2D SOF (Figure 4d). In sharp contrast, the UV-Vis spectra exhibited no change (Figure
S6, Supporting Information) when the mixture was kept in dark for 12 h, suggesting the
reversible formation and dissociation of the 2D SOF was induced by light. In addition, the
absorbance of the SOF at 345 nm showed periodic variations in four cycles, indicating the
recyclable and stable photo-responsiveness of this system (Figure 4b). The successful
preparation of the reversible light-responsive 2D SOF greatly promotes the development of
stimuli-responsive 2D device and lays a foundation for structuring advanced materials.
Interestingly, trans-cis isomerism of azobenzene derivatives can deplete the excited
energy, resulting in no fluorescence emissions.[34] On this basis, AZO could serve as an
efficient quencher in this system. As shown in Figure 5a, trans-AZO and cis-AZO can both
quench the fluorescence of TPE-4MV without CB[8]. The greater spectra overlap between the
absorption of cis-AZO and the emission of TPE-4MV resulted in far stronger quenching
ability of cis-AZO than that of trans-AZO (Figure 5b and 5d).[48] On the other hand, after
forming the host-guest complex, trans-AZO exhibited higher energy transfer efficiency and
stronger quenching effect since it was much closer to TPE-4MV in the cavity of CB[8].[49]
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Therefore, during the reversible formation and dissociation of the SOF, no fluorescence was
observed (Figure S7, Supporting Information), suggesting that the trans-cis isomerism of
AZO does not interfere with subsequent fluorescence recovery.
Figure 5. (a) Fluorescence spectra (λex=410 nm, λem=520 nm; slid width: ex=5 nm, em=10
nm) of TPE-4MV (0.1 mM), TPE-4MV (0.1 mM) and trans-AZO (0.2 mM), TPE-4MV (0.1
mM) and cis-AZO (0.2 mM). (b) Normalized emission spectrum of TPE-4MV and absorption
spectrum of trans-AZO. (c) Fluorescence spectra of TPE-4MV (0.1 mM) and SOF (0.1 mM)
after irradiation with UV light (365 nm) and visible light (450 nm) in PBS buffer (pH=7.4).
(d) Normalized emission spectrum of TPE-4MV and absorption spectrum of cis-AZO.
As an alternative to light-mediated isomerization, the reduction of nitrogen double bond
has been another stimulus of the system to restore fluorescence. Azoreductase, widely found
in most bacteria, can reduce azobenzene groups to anilines with high efficiency and
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selectivity.[50] Hence, it is expected that the 2D SOF would be an excellent fluorescent probe
to detect azoreductase activity in bacteria. To imitate physiological environment of bacteria,
fluorescence variation of the 2D SOF was studied in PBS buffer. In Figure 5c, the
fluorescence quenching of the SOF was affirmed as expected. Then sodium dithionite (SDT),
as an effective and biocompatible chemical substitute for azoreductase, was introduced to
reduce nitrogen double bond.[51] The kinetics of the reduction reaction were measured by UV-
Vis spectra (Figure 6a) and fluorescence spectra (Figure 6b). The placid absorbance of the
SOF at 345 nm showed a sharp decline after the addition of SDT (20 equiv.) and the light
yellow color of the SOF disappeared in 15 min, all of which indicated the reduction of AZO.
Meanwhile, the reduction of AZO resulted in a fast-speed fluorescence recovery of the
system. This process proved that the change of fluorescence intensity of the 2D SOF was
stimulated by the reduction of nitrogen double bond.
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Figure 6. (a) The absorbance at 345 nm and (b) the fluorescence intensity (λex=410 nm, λem=520
nm; slid width: ex=5 nm, em=10 nm) at 520 nm of 2D SOF as a function of time following
addition of SDT (20 equiv.) in PBS buffer (pH=7.4). Inset: absorbance spectra/fluorescence
spectra and photos of the SOF before and after addition of SDT in PBS buffer. (c) Fluorescence
responses of the SOF (the concentration of TPE-4MV, CB[8], and AZO in it was 0.1 mM, 0.4
mM, and 0.2 mM, respectively) in the presence of interfering species: 1 (blank); 2 (Na2S2O4, 2
mM); 3 (Escherichia coli); 4 (NaCl, 10 mM); 5 (KCl, 10 mM); 6 (CaCl2, 10 mM); 7 (MgCl2,
10 mM); 8 (cysteine, 1 mM); 9 (dithiothreitol, 1 mM); 10 (glutamic acid, 1 mM); 11 (arginine,
10 μM); 12 (H2O2, 10 μM); 13 (BSA, 100 μM). Experimental conditions: PBS buffer, pH=7.4,
37 °C. (d) Fluorescence emission time-course of the SOF in Escherichia coli culture medium.
Inset: photos of the Escherichia coli culture medium with or without the SOF under UV
illumination (365 nm).
Based on the above results, the 2D SOF, as the fluorescent probe to detect azoreductase
activity, was mixed into Luria-Bertani culture media. After the growth of Escherichia coli,
culture media showed a recovery of fluorescence as shown in the photos (Figure 6d), while
the blank control group did not change. Azoreductase activity can not only track the
proliferation of Escherichia coli, but also real-time monitor the growth process. The
fluorescence intensity of 2D SOF with the growth of Escherichia coli was shown in Figure
6d. The extreme rise of fluorescence intensity was consistent with the logarithmic growth
period of Escherichia coli from 1 h to 5 h. Then fluorescence intensity of the solution
increased gently because the growth of Escherichia coli has become stable.[52-53] Certainly,
potential interference factors, such as inorganic salts (NaCl, KCl, CaCl2 and MgCl2), biothiols
(cysteine and dithiothreitol), glutamic acid, arginine, reactive oxygen species (H2O2), and
bovine serum albumin (BSA), all have been estimated under the same conditions before
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actual testing (Figure 6c). The fluorescence intensity of 2D SOF hardly changed in these
interferences, indicating the selectivity of the SOF to azoreductase. The 2D SOF has exhibited
the excellent performance of being a fluorescence probe, detecting azoreductase activity and
real-time monitoring the growth process of Escherichia coli.
Conclusions
In conclusion, we have demonstrated that a 2D SOF can be constructed in aqueous
solutions through the formation of supramolecular stimuli-responsive host-guest complexes of
TPE-4MV, CB[8], and AZO. The reversible formation and dissociation of the 2D SOF was
induced by light, a stable, accurate, and long-range external stimulus, via the trans-cis
isomerization of AZO, rather than by harsh conditions and extra chemicals. Moreover, the 2D
SOF showed a “Turn-On” fluorescence response to the reduction of nitrogen double bond.
High selectivity to azoreductase against other bio-relevant interferences and the ability of
real-time monitoring the growth process of Escherichia coli made the SOF an excellent and
effective fluorescent probe. The 2D SOF provides a new direction for the construction of
chemical and biological devices with good stimulus responsiveness and easy manipulation.
Experimental Section
General Information
All the reagents and solvents were purchased from commercial sources and used as received
unless otherwise noted. Ultrapure water, purifying by Experimental Water System (Lab-UV-
20), was used in the relevant experiments. 1H NMR spectra were recorded on a Bruker
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AVANCE III 300 MHz NMR spectrometer at 298 K. 13C NMR spectra were measured on a
Bruker Digital Avance III HD 400 WB NMR spectrometer at ambient temperature with a
magic angle spinning rate of 7.0 kHz. SEM images were shot at 35 kV on a Hitachi SU8020
electron microscope operating. SAXD patterns were recorded on a Rigaku Smartlab X-ray
diffractometer. UV-Vis spectra were recorded on a Shimadzu UV-2550 instrument.
Fluorescence spectra were recorded on a Shimadzu RF-5301PC spectrometer. AFM image
was taken with keysight SPM6500. Precursors 1,1,2,2-tetrakis(4-(4-
bromobutoxy)phenyl)ethene (T2), (E)-1-(4-(4-bromobutoxy)phenyl)-2-phenyldiazene (A2),
and CB[8] were synthesized according to literature procedures.[54-58]
Synthesis of TPE-4MV: Compound T2 (1.87 g, 2 mmol) and 1-methyl-[4,4'-bipyridin]-1-
ium iodide (2.98 g, 10 mmol) were added into a flask. Then MeCN (50 mL) was added at
room temperature. Then the mixture was heated at reflux for 72 h. After cooling to room
temperature, the precipitate was filtered and washed with MeCN and DCM to result in a red
product (2.68 g, yield: 63%). 1H NMR (DMSO-d6, 300 MHz, δ): 9.45-9.43 (d, 8H), 9.32-9.30
(d, 8H), 8.83-8.77 (m, 16H), 6.84-6.83 (d, 8H), 6.71-6.69 (d, 8H), 4.80-4.75 (m, 8H), 4.45 (s,
12H), 3.96-3.92 (m, 8H), 2.16-2.10 (m, 8H), 1.78-1.71 (m, 8H). 13C NMR (126 MHz, CDCl3,
298K) δ 150.79, 149.78, 147.22, 146.32, 137.88, 133.06, 128.02, 127.95, 127.62, 127.51,
114.65, 67.97, 62.52, 37.53, 28.65, 26.22. HRMS (ESI): calculated for [M]3+
[C86H92Br4IN8O4]3+: 582.4326, found: 582.4364.
Real-time Detection of Azoreductase Produced by Escherichia Coli
The Luria-Bertani (LB) culture medium was prepared by bacto-tryptone (2 g), bactoyeast
extract (1 g) and NaCl (2 g) in water (200 mL), and the pH was adjusted to 7.4 using NaOH
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(1 M). The Escherichia coli was first grown at 37 °C in LB culture media. Then, the bacterial
colonies were added into 200 mL of fresh LB culture media containing the 2D SOF material.
The Escherichia coli was cultured in Luria-Bertani broth at 37 °C and taken for different
periods of time (0-12 h).[60]
Conflicts of interest
There are no conflicts to declare.
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements
We thank the National Natural Science Foundation of China (51673084 and 21871108), the
Jilin Province-University Cooperative Construction Project–Special Funds for New Materials
(SXGJSF2017-3), and the Jilin University Talents Cultivation Program for financial support.
Received: ((will be filled in by the editorial staff))
Revised: ((will be filled in by the editorial staff))
Published online: ((will be filled in by the editorial staff))
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19
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10.1002/chem.201904443
Chemistry - A European Journal
Dual-stimuli responsive two-dimensional (2D) supramolecular organic framework is
constructed in aqueous solution via self-assembly of cucurbit[8]uril and guests. The
reversible formation and dissociation of the 2D SOF is realized through light-mediated
isomerization of azobenzene, which facilitates the real-time detection of the azoreductase
activity in Escherichia Coli by the enzymatic reduction.
Keywords: cucurbit[n]uril, fluorescent probe, photoisomerization, stimuli-responsive system,
supramolecular organic framework
Hao Zhang, Feng Liang,* and Ying-Wei Yang*
Dual-Stimuli Responsive 2D Supramolecular Organic Framework for the Detection of
Azoreductase Activity
20
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